Neuropeptide Y (NPY) is a 36 amino acid alpha-amidated peptide widely found throughout the nervous system. Coexistence of neuroactive peptides and classical transmitters in neurons has been frequently observed. The proposed studies examine the mechanisms of co-regulation of synthesis, secretion, and storage of multiple co-localized neuroactive substances. The model system chosen for these studies is rat superior cervical ganglion (SCG) culture. SCG neurons produce norepinephrine (NE), acetylcholine (ACh), and NPY. The initial aim of this proposal is to investigate the regulation of NPY biosynthesis. Studies will detail the synthetic pathway from pro-npy to NPY and other product peptides in SCG neurons. Proposed co- and post- translational modifications of pro-NPY including amidation and phosphorylation will be examined. NPY mRNA will be measured by Northern gel analysis. NPY-containing SCG neurons will be identified by in situ hybridization and regulation of synthesis within this sub-population of cells will be studied. The second major goal is to examine neuronal co-regulation of coexisting neuroactive substances in SCG. Studies will explore the effect of known regulatory factors on the contents and synthesis of NPY, NE, and ACh. In particular, the effect of changes in neuronal activity and in the culture medium on transmitter and peptide contents, synthetic rates, and NPY mRNA levels will be determined. The SCG model system also enables study of two related biosynthetic enzymes, peptidyl-glycine alpha-amidating monooxygenase (PAM) and dopamine beta-hydroxylase (DBH) and enables comparison of PAM activity with amidated NPY under various conditions. NPY and NE neurons will also be identified by immunochemistry and NPY neurons by in situ hybridization so that the control of sub-populations of NPY-containing neurons in SCG can be investigated. NPY is a potent constrictor of vascular smooth muscle. In addition the localization of NPY in sympathetic ganglion and other brainstem sites suggests a role in neuronal control of blood pressure and autonomic function. It is expected that these studies will provide further insights into the regulation of NPY in neurons and more broadly into the co- regulation of coexisting peptides and transmitters.